This renewal application extends this group's previous interest in evaluating the signal transduction pathways initiated during T-cell activation. During the previous funding period, molecular strategies were successfully employed to assess the differential roles of src-family and syk-family protein tyrosine kinases (PTK) in TCR-initiated signaling. Preliminary data are provided in this application to suggest that a signal transducing protein, Sam68, also plays a critical role in proximal TCR-initiated signaling. Specifically, the data suggest that the recruitment of Sam68 by the SH2 and SH3 domains of lck is required for the downstream effector functions of ZAP-70. Expression of a "dominant negative" form of Sam68 disrupts signal transduction from the TCR. For this application, the investigators will: (1) Identify elements of Sam68 which interact with downstream effectors; (2) Create ES cell lines bearing a homozygous targeted disruption of the Sam68 gene and study the phenotype of T-cells resulting from injection of the ES cells into RAG2-/- blastocyst; (3) Use biochemical and genetic approaches to identify downstream effectors which interact with Sam68; (4) Identify the elements of lck which are required for signal transduction steps immediately upstream of the TCR phosphorylation; and (5) Identify upstream effectors which interact with the Lck unique region.